Head and neck cancer is often treated with surgical excision as the first therapeutic method. Local disease control and eventual survival are closely associated with achievement of clear surgical margins. However, many patients suffer recurrence of tumor despite surgery with histologically clear margins. Over a decade ago, we showed that molecular evaluation for the presence of rare tumor cells in histologically clear resection margins was associated with a high risk of local recurrence. However, this approach has not been adopted in routine clinical practice because of the technically challenging nature of the molecular probing of margin DNA. The overall goal of this proposal is to complete the steps necessary to demonstrate that molecular assessment of surgical resection margins is a valuable and feasible tool for generalized clinical use in the management of head and neck mucosal malignancies. The platform for this endeavor is a high quality clinical sample and corresponding data set from the ECOG E4393/RTOG 9614 head and neck margin study of which Dr. Koch is PI. The project has three specific aims: (1) Verification of a panel of tumor-specific molecular markers as robust and universal for HNSCC surgical resection margin analysis. Hypermethylation of four genes identified in the Hopkins head and neck SPORE shows great promise for this panel. (2) Molecular margin analysis for prediction of outcome: Rigorous assessment of the utility of molecular margin analysis using this large, well characterized cohort of HNSCC tumors and margins will be followed by a search for correlates between molecular and clinical outcome. (3) Determine the clinical correlates of tumor-specific molecular alterations. This last aim builds on our recent successful identification of a high risk subpopulation of mutations of TP53 in this cohort of HNSCC tumors capitalizing on its large size and mature outcome information. With the successful accomplishment of these steps, adoption of the practice of molecular margin analysis by cancer centers with a substantial volume of HNSCC cases is anticipated.